Fernanda de Oliveira Bustamante

Functional repetitive sequences and fragile sites in chromosomes of Lolium perenne L.

Lolium perenne is considered a high-quality forage widely used in temperate regions to meet the shortage of forage during the winter. In this species, some peculiarities related to cytogenetic aspects have already been described, as the variability in number and position of 45S ribosomal DNA (rDNA) sites and the expression of fragile sites, which require further studies to support the understanding of their causes and consequences. In this way, this study aimed to evaluate the relationship between the expression of fragile sites and functional repetitive sequences (rDNA and telomeric) in chromosomes of diploid and polyploid cultivars of L. perenne. The techniques of FISH, Ag-NOR and fluorescence banding were used to assess the distribution of sites of 45S rDNA, 5S, telomeric sequences, and the transcriptional activity of the 45S ribosomal genes and the distribution of AT- and/or GC-rich sequences in L. perenne, respectively. There was variability in the number and location of 45S rDNA sites, which was not observed for 5S rDNA sites. One of the genotypes showed two 45S rDNA sites on the same chromosome, located in different chromosome arms. Breaks and gaps were found in 45S rDNA sites in most metaphases evaluated for both cultivars. Telomeric sequences were not detected at the end of the chromosomal fragments corresponding to the location of breaks at 45S sites. Apparently, the transcriptional activity was modified in fragile sites. Variation in the number and size of nucleoli, nucleolar fusions and dissociations were observed. All CMA+ bands were colocalized with the 45S sites.

Meiotic behavior in early and recent duplicated hexaploid hybrids of napier grass (Pennisetum purpureum) and pearl millet (Pennisetum glaucum)

The production of hybrids between napier grass (Pennisetum purpureum, 2n = 4x = 28, genomes A′A′BB) and pearl millet (P. glaucum, 2n = 2x = 14, genome AA) results in sterile triploid plants. Such sterility of the triploid hybrid creates a hurdle for napier grass breeding programs, since it prevents propagation through seeds. Fertility can be restored by means of chromosome duplication. In most cases, these duplicated Pennisetum hybrids are mixoploids. However, we do not know if there are differences between the meiotic behavior of Pennisetum hybrids between napier grass and pearl millet which were duplicated early on and those duplicated more recently. This work evaluated the meiotic behavior and the pollen viability of American and Brazilian hexaploid hybrids, which differ by about 25 years in duplication time. In all analyzed hybrids, irregularities were observed from the initial phases of meiosis to pollen grain production, independently from the time elapsed for the duplication induction. The most frequent meiotic abnormalities are related to chromosome segregation due to irregular pairing. These irregularities will directly affect the viability and the size of the pollen grains.

Morphology and pollen viability of Lolium multiflorum Lam.

O estudo e a caracterizacao do grao de polen sao fundamentais para diferentes areas, em especial, para a taxonomia, o melhoramento genetico, a filogenia e a paleobotânica. Para genotipos de Lolium multiflorum Lam., naturalizados no Brasil, cultivares introduzidas ou populacoes de melhoramento, diploides e poliploides, nao existem relatos sobre a caracterizacao morfopolinica. Nesse trabalho foram analisados dez gentotipos de azevem anual (L. multiflorum). A viabilidade dos graos de polen foi verificada por coloracao com os corantes carmim propionico e Alexander e a morfologia por meio da tecnica de acetolise, sendo obtidas medidas do eixo polar (P), do diâmetro equatorial (E), e da espessura da exina, e analise dos graos de polen por microscopia eletronica de varredura (MEV). Todos os genotipos avaliados apresentaram alta taxa de viabilidade polinica (>89%) para os dois corantes utilizados. Houve diferenca entre os genotipos para os caracteres quantitativos eixo polar, diâmetro equatorial na vista equatorial, exina, endoexina, ectoexina e razao P/E. Os graos de polen foram caracterizados como pequenos, monoaperturados, com abertura nao proeminente e com âmbito circular. As diferencas morfometricas entre os graos-de-polen podem auxiliar na distincao entre os mesmos e, posteriormente, serem utilizadas para comparacao entre os niveis de ploidia, auxiliando, assim, nos programas de melhoramento da especie.

Identification of haploid maize by flow cytometry, morphological and molecular markers

The development of homozygous breeding lines in maize may be accelerated through the use of haploids. Thus, the obtaining and prior identification of haploids generated by the haploid inducer lines is an important factor. The purpose of this study was to identify haploids by flow cytometry and to correlate the nuclear DNA content to the morphological and morphometric traits of the seeds that gave rise to them. In addition, molecular markers were used to confirm the androgenetic nature of the haploid. The seeds obtained were derived from the cross between the inbred line W23 and the commercial hybrid P30F90. Among these seeds, a group was selected, putative haploids, whose embryo was white and the pericarp purplish. This group, consisting of 330 seeds, was characterized based on seed morphology, seed morphometry and nuclear DNA content. Flow cytometry analyses identified four haploids, and all of them were small size plants and had brittle leaves. The weight, length, thickness and width of the haploid seeds were very variable indicating that morphometric traits do not constitute reliable data for visual selection of haploid seeds. Based on results, the inbred line W23 induced haploid maize even under tropical conditions. Microsatellite molecular markers (SSR) proved to be efficient, confirming the androgenetic trait of the haploids.

Genomic behavior of hybrid combinations between elephant grass and pearl millet

Abstract – The objective of this work was to evaluate the genomic behavior of hybrid combinations between elephant grass ( Pennisetum purpureum ) and pearl millet ( P. glaucum ). Tetraploid (AAA’B) and pentaploid (AA’A’BB) chromosome races resulting from the backcross of the hexaploid hybrid to its parents elephant grass (A’A’BB) and pearl millet (AA) were analyzed as to chromosome number and DNA content. Genotypes of elephant grass, millet, and triploid and hexaploid induced hybrids were compared. Pentaploid and tetraploid genomic combinations showed high level of mixoploidy, in discordance with the expected somatic chromosome set. The pentaploid chromosome number ranged from 20 to 34, and the tetraploid chromosome number from 16 to 28. Chromosome number variation was higher in pentaploid genomic combinations than in tetraploid, and mixoploidy was observed among hexaploids. Genomic combinations 4x and 5x are mixoploid, and the variation of chromosome number within chromosomal race 5x is greater than in 4x.Index terms:

Variation of karyotype and nuclear DNA content among four species of Plectranthus L’ Héritier, 1788 (Lamiaceae) from Brazil

Abstract Plectranthus is a genus which includes species of ornamental and medicinal potential. It faces taxonomic problems due to aggregating species previously belonging to the genus Coleus, a fact that has contributed to the existence of various synonymies. The species Plectranthus amboinicus, Plectranthus barbatus, Plectranthus grandis and Plectranthus neochilus are included in this context. Some authors consider Plectranthus barbatus and Plectranthus grandis as synonyms. The present work was carried out with the aim of comparing plants of the above-mentioned species, originating from different localities in Brazil, with regards to chromosome number and karyotypic morphology, correlated to the nuclear DNA content. There was no variation in chromosome number among plants of the same species. Plectranthus amboinicus was the only species to exhibit 2n=34, whereas the others had 2n=30. No karyotypic differences were found among the plants of each species, except for Plectranthus barbatus. The plants of the Plectranthus species revealed little coincidence between chromosome pairs. The nuclear DNA content allowed grouping Plectranthus amboinicus and Plectranthus neochilus, with the highest mean values, and Plectranthus grandis and Plectranthus barbatus with the lowest ones. Differences in DNA amount among the plants were identified only for Plectranthus barbatus. These results allow the inference that the populations of Plectranthus amboinicus and Plectranthus neochilus present coincident karyotypes among their plants, and Plectranthus grandis is probably a synonym of Plectranthus barbatus.

In vivo HAPLOID INDUCTION AND EFFICIENCY OF TWO CHROMOSOME DUPLICATION PROTOCOLS IN TROPICAL MAIZE

Artificial chromosome duplication is one of the most important process in the attainment of doubled haploids in maize. This study aimed to evaluate the induction ability of the inducer line KEMS in a tropical climate and test the efficiency of the R1-Navajo marker by flow cytometry to evaluate two chromosome duplication protocols and analyze the development of the doubled haploids in the field. To accomplish this goal, four genotypes (F1 and F2 generations) were crossed with the haploid inducer line KEMS. The seeds obtained were selected using the R1-Navajo marker and subject to two chromosome duplication protocols. Duplication was confirmed using flow cytometry. The percentages of self-fertilized plants after duplication as well as the quantities of doubled haploid seeds obtained after the self-fertilization processes were analyzed. It was observed that the germplasm influences haploid induction but not the duplication rates of the tested protocols. Protocol 2 was more efficient for the duplication of haploids, in the percentage of self-fertilized plants after duplication, and in the attainment of doubled haploid lines. Moreover, the haploid inducer line KEMS can produce haploids in a tropical climate. Other markers, in addition to the R1-Navajo system, should be used in the selection of haploid seeds.